Virology

• Clinical Disease: In clinical disease outbreaks of suspected IHNV in sockeye salmon, 10 moribund or freshly dead fish are sufficient to isolate the virus for a confirmed diagnosis. In other salmonid species, 60 moribund fish may be required to establish an etiology. For alevins, fry, and fingerlings, whole fish should be sent by following instructions given above under finfish bacteriology. Also, enclose 10 additional moribund fry per lot, 5 per bag or the equivalent number to equal 1 g if very small fish. Do not add water.
• Broodstock and Disease History Examination: For establishing a disease history in adult fish or in broodstock screening, 60 samples from adult fish will be required. Samples of choice are from spawning or postspawning female fish consisting of ovarian fluids collected from each fish and shipped in separate disposable centrifuge tubes with snap caps. When required, samples from spawning or postspawning males should consist of 0.5 g each anterior and posterior portions of kidney and whole spleen from each fish, aseptically removed and pooled in individual sealed 2 ounce white-labeled plastic Whirl- Pak Tissues from more than one fish should not be combined in one bag. All tissues and fluids for virus assays should be shipped to the FPS on blue ice (4 degree C) but never frozen. Freezing at low temperatures and subsequent thawing can inactivate IHNV, producing lower titers, which in some samples may be too low to detect routinely. Virus samples on blue ice should be sent to the FPS lab as soon as possible within 72 hours of collection.

These sampling procedures are applicable to assays for other finfish viruses should the need arise.

1. Ovarian fluids for virology testing: Obtain instructions from the lab staff regarding whether you should take ovarian fluids from ripe fish used in the egg take or from postspawning fish. Disinfect the external ventral surface and wipe dry with paper towels. For postspawners, partially strip a single fish's ovarian fluids into a paper cup (recommend 4 oz pleated cups but paper drink cups can be used), avoiding the extrusion of blood, fecal material or nematode worms if present. For ripe fish, you may either extrude a small amount of fluid prior to taking eggs or pour fluid off the eggs. Two ml of fluid are adequate for ripe fish, but 3-5 ml should be obtained if sampling postspawners in case there is a need to filter the samples. Crimp edges of the cup to form a spout and pour fluid into a 10 ml centrifuge tube with cap, "straining out" any eggs. Avoid contaminating the rim with your hands. Discard the cup after each fish. Do not provide more than 5 ml of ovarian fluid. Cap the tube tightly making sure that the cap is not improperly seated. Place tubes in a rack in a plastic Ziploc bag labeled with stock of fish and species, sampling location, date, fish life stage, and number of samples. Place upright in a cooler with cold packs. Do not freeze. If samples cannot be shipped within 72 hours they may be kept on ice up to 6 days if not grossly contaminated. Otherwise they must be frozen.
2. Tissue samples from males for virology testing: Disinfect the external ventral surface and either rinse with clean, pathogen-free water or wipe dry with paper towels. Carefully cut open fish, taking care not to cut the gastrointestinal tract which would contaminate tissues with bacterial flora. Aseptically remove the spleen and anterior and posterior portions of the kidney, each about 0.5 g in size (size of thumbnail) and place into a single 2 oz white-labeled plastic Whirl-Pak bag using a spoon, knife or forceps. Seal each bag and keep cool (4 degree C).  Between the sampling of each fish, clean dissecting utensils with 3% iodine and dry with a clean paper towel. Organic matter will affect the working ability of the disinfectant, so any tissue should be wiped off utensils with a separate paper towel prior to disinfection. Wooden tongue depressors, discarded between samples, eliminate the need for disinfection. Disinfect hands between the sampling of each fish. Rubber gloves should be worn to reduce human contact with disinfectant. When sampling is done, place all sealed sample bags in a large plastic bag. Label the bag with the number of samples, stock of fish and species, sample location, fish life stage and date. Place in a cooler on ice or a cold pack and transport within 3 days. Do not freeze. Make sure you include a Sample Submission Form with each shipment.